I am trying to measure phosphorylation of lamin A subunits by 2D-PAGE. I resuspend Vero cells in Rehydration buffer (which contains 8M urea) and sonicate them. I run this cell lysate sample on an IPG strip, add the strip to the top of an SDS-PAGE gel, and do everything as I would a Western blot. I am using a polyclonal antibody that I know works well with Western blots, but I get absolutely no signal for the 2D-PAGE. There is not a problem with the sample because blotting with another antibody does work.
Thinking there could be a problem with lamin degradation in 8M urea, I resuspended half my sample in RIPA buffer and half in the Rehydration buffer. I added SDS sample buffer to each of these samples did a Western blot for lamin A. The sample in RIPA buffer had a signal, but the Rehydraton buffer was blank. I blotted for another protein and there was signal in both the RIPA buffer and the Rehydration buffer sample.
Several papers have shown the ability to do 2D-PAGE for lamin A with a cell lysate sample in Rehydration buffer, so I don't really think lamin A is being degraded. Does anyone know if urea can denature an epitope for a polyclonal antibody that works for Western blot? Any other ideas as to why I would get a signal that a sample in RIPA buffer but not an 8M urea buffer?
Urea can induce deamination of protein. If your protein was in urea buffer and was stored for too time, buffer can dissociate in cianate and tiocianate ions, and these can induce deamination and loss of functional properties.
Please see the product information about Urea solution:
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Datasheet/u4883dat.pdf
I wondered if you sonicated your sample too much which resulted in overheating your solution and unfavorable alterations to your samples.
You might want to try a different rehydration buffer, like the ones shown in the papers you mentioned. They may have different compositions, good to preserve your target.
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